CN108387728A - A kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion and detection method - Google Patents

Abstract

The present invention relates to the field of chemical detection, in particular to a triazophos bionic enzyme-linked immunosorbent detection kit and a detection method; the kit includes: a molecularly imprinted polymer membrane enzyme-labeled plate and a triazophos enzyme-labeled antigen ; The preparation is obtained by the following method: using triadimefon as a template molecule, performing prepolymerization with functional monomers, crosslinking agents, and initiators in an inert solvent to obtain a prepolymerization solution; adding the prepolymerization solution to a microtiter plate In , the template molecules were eluted after vacuum drying and polymerization. The triazophos adsorption functional material provided by the invention has high stability, long service life and strong ability to resist harsh environments, and overcomes the shortcomings of traditional biological antibodies such as long preparation period, easy inactivation, and high cost.

Description

A kind of triazophos bionic ELISA detection kit and detection method

technical field

The invention relates to the field of chemical detection, in particular to a triazophos bionic ELISA detection kit and a detection method.

Background technique

Triazophos is a moderately poisonous, broad-spectrum organophosphorus insecticide, which has strong contact and stomach poisoning effects. After entering the human body with food, it will cause a large amount of acetylcholine to accumulate at the junction of neuroeffectors, and mushrooms will occur. Alkali-like, nicotine-like and central nervous system symptoms, since December 31, 2016, the use of triazophos on vegetables is prohibited.

At present, the routine detection methods of triazophos include gas chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, etc., but these methods are complicated in pretreatment, high in cost and slow in detection speed, and are not suitable for on-site real-time detection. Immunoassay has high specificity, high sensitivity, and high selectivity unmatched by conventional physical and chemical analysis, and plays a pivotal role in clinical testing and laboratory research. However, traditional biological antibodies also show many shortcomings. For example, small molecule compounds must synthesize haptens to be immunogenic, the preparation process is cumbersome, the cycle is long, and animals need to be sacrificed, and the physical and chemical properties of biological antibodies are unstable, and storage and use conditions are strict, etc. . Therefore, it is of great practical significance to design and synthesize biomimetic antibodies with high specificity, good stability and low cost that can be compared with biological antibodies. With the development of polymer chemistry and the emergence of molecular imprinting technology, molecularly imprinted polymers have become ideal materials for biomimetic antibodies. MIP is a synthetic polymer material with specific recognition sites, which has many advantages over biological antibodies. Therefore, the combination of molecular imprinting technology and enzyme-linked immunosorbent assay, the preparation of triazophos molecular imprinting membrane, and the establishment of bionic enzyme-linked immunosorbent assay technology for the rapid detection of triazophos pesticide residues provide a reference for the study of trace detection of pollutants. The monitoring of triazophos pollution in agricultural products provides technical support and is also an important supplement to the current research on rapid analysis and detection technology, which will promote the development of rapid detection technology for pesticide residues.

In view of this, the present invention is proposed.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the traditional triazophos detection method, such as long detection time, expensive equipment, and difficult preparation of specific antibodies, to provide a rapid detection kit and method for triazophos.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

The invention relates to a triazophos bionic ELISA detection kit, comprising:

Molecularly imprinted polymer membrane microtiter plate and triazophos enzyme-labeled antigen;

The preparation is obtained by the following method: using triadimefon as a template molecule, performing prepolymerization with functional monomers, crosslinking agents, and initiators in an inert solvent to obtain a prepolymerization solution; adding the prepolymerization solution to a microtiter plate , template molecules can be eluted after vacuum drying and polymerization.

According to one aspect of the present invention, the present invention also relates to a method for using the above-mentioned kit to detect triazophos biomimetic ELISA, comprising:

Set blank control wells, negative control wells and detection wells on the molecularly imprinted polymer membrane enzyme plate;

Adding triazophos enzyme-labeled antigen diluent, and triazophos standard solution or sample extraction solution into the detection well, and the triazophos standard solution or sample extraction solution added in different wells are diluted in gradient;

Add triazophos enzyme-labeled antigen diluent and buffer to the negative control well;

Wherein, the triazophos enzyme-labeled antigen diluent is obtained by diluting the triazophos enzyme-labeled antigen standard solution with a buffer;

Add the buffer solution to the blank control well;

Incubate at room temperature for 50-70 minutes after adding the wells according to the above method, add substrate solution to each well after washing the plate, and terminate the reaction after incubating at room temperature for 30-60 minutes; read the absorbance of each well with a microplate reader and calculate the inhibition according to the following formula Rate:

IC%=[1-(A _sample -A _blank )÷(A _control -A _blank )]×100;

In the formula:

IC% - the inhibition rate of triazophos on the antigen-antibody binding reaction;

A _control —the average absorbance value of the negative control well;

A _sample ——the average absorbance value of triazophos standard solution or sample extract in the detection hole;

A _blank —the average absorbance value of the blank control well;

Take the logarithmic value of the concentration of the gradient dilution of the triazophos standard solution or the concentration of the gradient dilution of the sample extract as the abscissa, and the corresponding percentage of inhibition rate as the ordinate, and draw the standard curve respectively;

The absorbance value corresponding to the sample extract is substituted into the standard curve to obtain the concentration of the sample extract.

Compared with prior art, the beneficial effect of the present invention is:

(1) The triazophos adsorption functional material provided by the present invention has high selectivity, and the use of virtual templates can solve the template leakage problem, and can replace biological antibodies in ELISA; the adsorption functional material is prepared by chemical methods, It has high stability, long service life and strong ability to resist harsh environments, and overcomes the shortcomings of traditional biological antibodies such as long preparation period, easy inactivation, and high cost.

(2) The present invention combines molecular imprinting technology with enzyme-linked immunosorbent technology to establish a bionic ELISA detection method with high sensitivity to triazophos. The minimum detection limit of this method is 7μg·L ^-1 , which is much lower than the maximum residue limit and can meet the detection needs. And the analysis time is greatly shortened (1.0h shorter than the traditional biological immunoassay), which is suitable for rapid detection of triazophos.

(3) The biomimetic antibody prepared by the present invention can be reused more than 50 times, and the cost is greatly reduced; and the pretreatment is simple, the sensitivity is high, and the experimental operation is simple, which is suitable for the rapid detection of triazophos in various foods.

Detailed ways

The invention relates to a triazophos bionic ELISA detection kit, comprising:

Molecularly imprinted polymer membrane microtiter plate and triazophos enzyme-labeled antigen;

The preparation is obtained by the following method: using triadimefon as a template molecule, performing prepolymerization with functional monomers, crosslinking agents, and initiators in an inert solvent to obtain a prepolymerization solution; adding the prepolymerization solution to a microtiter plate , template molecules can be eluted after vacuum drying and polymerization.

Preferably, in the kit as described above, the functional monomer is methacrylic acid, the crosslinking agent is trimethoxypropyl trimethacrylate, and the template molecule: functional monomer: crosslinking agent The molar ratio is 1:5～7:2～4;

More preferably, the molar ratio is 1:6:3.

Preferably, in the above-mentioned kit, the inert solvent is acetonitrile; more preferably, the template molecule: inert solvent = 1 mol: 20-30 ml; more preferably 1 mol: 25 ml.

Preferably, in the above-mentioned kit, the initiator is azobisisobutyronitrile; more preferably, the template molecule: initiator = 1 mol: 40-60 mg; more preferably 1 mol: 50 mg.

Preferably, in the above-mentioned kit, the conditions for the vacuum-drying polymerization are 30°C-50°C polymerization for 12h-24h under vacuum drying conditions;

More preferably, it is polymerized at 40° C. for 18 hours.

Preferably, in the kit as described above, the preparation method of the triazophos enzyme-labeled antigen comprises:

Synthesize the coated antigen from the triazophos hapten by the mixed acid anhydride method, and then couple the coated antigen to the enzyme protein at a molar ratio of 18 to 22:1, more preferably at a molar ratio of 20:1;

Preferably, the enzyme protein is horseradish peroxidase.

Preferably, in the above-mentioned kit, the step of synthesizing the coated antigen with the triazophos hapten by the mixed acid anhydride method specifically includes: mixing the triazophos hapten with N,N-dimethylformamide at a concentration of 0.2 mmol ～0.3mmol: 1ml was mixed to obtain a mixed solution, and then n-tributylamine and ethyl chloroformate were added to the mixed solution to react for 50min～70min, and then the obtained reaction solution was mixed with CBS buffer solution in which OVA was dissolved, and after dialysis instant;

More preferably, the step of synthesizing the coated antigen with the triazophos hapten by the mixed acid anhydride method specifically includes: mixing the triazophos hapten with N,N-dimethylformamide at a ratio of 0.25mmol: 1ml to obtain For the mixed solution, n-tributylamine and ethyl chloroformate were added to the mixed solution to react for 60 minutes, and then the obtained reaction solution was mixed with a CBS buffer solution in which OVA was dissolved, and obtained after dialysis.

Preferably, the kit as described above further includes a buffer; more preferably, the buffer is a PBS solution.

Preferably, the kit as described above, said kit also includes a substrate solution;

The substrate liquid is configured from substrate A and substrate B;

The ingredients of the substrate A include: 8.0g/L-8.4g/L anhydrous sodium acetate, 2.0g/L-3.0g/L β-cyclodextrin and 140mg/L-160mg/L peroxide Hydrogen urea, pH=4.8～5.2;

The composition of the substrate B includes 3,3,5,5-tetramethylbenzidine: dimethyl sulfoxide in a ratio of 40 mg to 60 mg: 4 ml to 6 ml;

More preferably, the composition of the substrate A includes: 8.2g/L anhydrous sodium acetate, 2.5g/L β-cyclodextrin and 150mg/L urea hydrogen peroxide, pH=5.0;

The composition of the substrate B includes 3,3,5,5-tetramethylbenzidine:dimethylsulfoxide in a ratio of 50mg:5ml.

According to one aspect of the present invention, the present invention also relates to a method for using the above-mentioned kit to detect triazophos biomimetic ELISA, comprising:

Set blank control wells, negative control wells and detection wells on the molecularly imprinted polymer membrane enzyme plate;

Adding triazophos enzyme-labeled antigen diluent, and triazophos standard solution or sample extraction solution into the detection well, and the triazophos standard solution or sample extraction solution added in different wells are diluted in gradient;

Add triazophos enzyme-labeled antigen diluent and buffer to the negative control well;

Wherein, the triazophos enzyme-labeled antigen diluent is obtained by diluting the triazophos enzyme-labeled antigen standard solution with a buffer;

Add the buffer solution to the blank control well;

Incubate at room temperature for 50-70 minutes after adding the wells according to the above method, add substrate solution to each well after washing the plate, and terminate the reaction after incubating at room temperature for 30-60 minutes; read the absorbance of each well with a microplate reader and calculate the inhibition according to the following formula Rate:

IC%=[1-(A _sample -A _blank )÷(A _control -A _blank )]×100;

In the formula:

IC% - the inhibition rate of triazophos on the antigen-antibody binding reaction;

A _control —the average absorbance value of the negative control well;

A _sample ——the average absorbance value of triazophos standard solution or sample extract in the detection hole;

A _blank —the average absorbance value of the blank control well;

Take the logarithmic value of the concentration of the gradient dilution of the triazophos standard solution or the concentration of the gradient dilution of the sample extract as the abscissa, and the corresponding percentage of inhibition rate as the ordinate, and draw the standard curve respectively;

The absorbance value corresponding to the sample extract is substituted into the standard curve to obtain the concentration of the sample extract.

Preferably, as described above, the preparation method of the sample extract comprises:

Crush the sample to be tested, add buffer solution, ultrasonically extract, and filter.

Preferably, in the above-mentioned method, the ratio of the sample to be tested to the buffer solution is 1g-2g: 5ml-10ml.

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1

This embodiment provides a biomimetic immunoassay method for triazophosphatase labeling, comprising the following steps:

1. Synthesis of hapten:

(1) Synthesis of O-ethylthiophosphoryl dichloride (TZM-1)

Weigh 68g (about 0.4mol) of phosphorus trichloride (PSCl ₃ ) into a three-necked flask with a low temperature thermometer, cool it to -10~-5°C with an ice-salt water bath, and add anhydrous 55g (about 1.2mol) of ethanol, and the rate of addition is strictly controlled so that the temperature of the reaction solution is always not greater than 0°C. After the dropwise addition, the reaction was continued at 10°C for 2h. After the reaction was completed, wash the reaction solution (100ml×2) with (0±5)°C distilled water, separate the oil layer and dry it with anhydrous Na ₂ SO ₄ , and then distilled under reduced pressure with a water pump to collect fractions at 65°C to 75°C to obtain 51.8 g colorless transparent oily liquid (yield 72.3%, calculated as phosphorus trichloride).

(2) Synthesis of O-ethyl-O-[3-(1-phenyl-1,2,4-triazolyl)phosphoryl thiochloride (TZM-2)

Weigh 36g (about 0.2mol) of TZM-1 into a 250ml three-necked flask, and add about 16g (about 0.1mol) under stirring. 1-Phenyl-1,2,4-triadiazole, then 15ml TEA and 80ml DCM were added. After all the solids were dissolved, the solution was cooled to below 20°C with an ice-water bath, a small amount of catalyst was added and 55ml of 2mol/L NaOH aqueous solution was added dropwise, and the reaction was continued for 1h. After completion of the reaction, add 50ml of 5% NaOH ice solution, shake and separate the water layer, wash the oil layer with ice water until neutral, then dry over anhydrous Na2SO4, and concentrate under reduced pressure to obtain a small amount of brown oil, which is extracted with petroleum ether (50ml×2), and the extract was concentrated under reduced pressure to obtain 10.6g of yellow liquid (35% yield, calculated as triadimenol).

(3) Synthesis of triazophos hapten

Weigh 1.03g (about 10mmol) of 4-aminobutyric acid, dissolve it in 10ml NaOH solution (1mol/L), cool in an ice-water bath to 0-10°C, slowly add 1.51g (about 5mmol) to dissolve in 10ml dioxane Ring TZM-2, add a small amount of catalyst, and then dropwise add 10ml NaOH aqueous solution (1mol/L). Raise the temperature to 15-25°C and react for 4h. After the reaction is complete, add 50ml of water, then wash the reaction mixture with petroleum ether (40ml×2), discard the petroleum ether layer, adjust the pH of the aqueous phase to about 3 with 2mol/L HCL, and then extract with ethyl acetate (40ml×2) , the extract was washed with a small amount of water, dried over anhydrous Na2SO4, concentrated under reduced pressure, and the residue was sealed and stored at 4°C overnight, and a colorless product was precipitated. Recrystallize from ethyl acetate-petroleum ether, filter, and dry to obtain 0.52 g of white solid (THBu, yield 27%, based on intermediate TZM-2).

2. Preparation of enzyme-labeled antigen

Take 0.25 mmol of the hapten, dissolve it in 1 ml of DMF, add 60 μl of n-tributylamine and 30 μl of ethyl chloroformate under stirring, and stir at room temperature for 1 h. Take 300μl of the reaction solution and slowly add it dropwise to 6ml of CBS buffer solution (0.01mol/L) dissolved with 120mgOVA. After stirring the reaction at room temperature, put it into a dialysis bag, first dialyze with distilled water for 3 times, and then dialyze with 0.01mol/L of PBS 3d, change the dialysate 3-4 times a day. Take out aliquots and store in a -20°C refrigerator.

After the antigen is activated, it is coupled to the enzyme protein, and the concentration of the enzyme protein in the reaction solution is 10 mg/ml (the molar ratio of antigen to enzyme protein is 20:1, and the enzyme is horseradish peroxidase). After the reaction, an equal volume of glycerin was added, mixed evenly, aliquoted, and stored at -20°C.

The 5-fold PBS solution is composed of Na ₂ HPO ₄ 12H ₂ O: 68.80g, NaH ₂ PO ₄ 2H ₂ O: 8.97g and NaCl: 45.00g, and distilled water to 1L; Dilute 5 times PBS with double distilled water 5 times to get PBS solution.

3. Preparation of Molecularly Imprinted Polymer Membrane

Triadimefon (dissolved in 20ml of acetonitrile), methacrylic acid and trimethoxypropyl trimethacrylate were mixed according to the molar ratio of 1:6:3, then 40mg of azobisisobutyronitrile was added, and the reaction was stirred for 0.5h. Add 200 μl of the above mixed solution to each well of a 96-well microplate plate, polymerize at 50°C for 12 hours under vacuum drying conditions, elute with 8:1 (v/v) methanol/glacial acetic acid solution for 8 hours, and elute with methanol solution for 3 hours to remove three Zazolone was dried to obtain molecularly imprinted polymer membranes.

4. Establishment of multi-residue enzyme-labeled biomimetic immunoassay method

The imprinted polymer membrane prepared in step 3 was used as a biomimetic antibody; the enzyme-labeled antigen standard solution prepared in step 2 was diluted 5000 times with PBS solution as the enzyme-labeled diluent.

Set the first row of the 96-well ELISA plate as the blank group, and add only 200 μL of PBS solution to each well; the second row as the control group, add 100 μL of enzyme standard dilution solution and 100 μL of PBS solution to each well; Add triazophos standard solution or sample extract gradient dilution solution and 100 μL enzyme standard dilution solution respectively. After incubation at room temperature for 1 h, the plate was washed five times with phosphate Tween buffered solution PBST. Add 100-200 μL of substrate solution to each well of the 96-well ELISA plate, and react for 30-60 minutes at room temperature. Add 50-100 μL of 1.25mol/L sulfuric acid to each well to terminate the reaction; read the absorbance value A of the 1-8 row of the 96-well microplate plate with a microplate reader, and calculate the inhibition rate respectively;

Calculate the inhibitory rate value of different concentrations of triazophos to the antigen-antibody binding reaction according to the following formula:

IC%=[1-(A _sample -A _blank )÷(A _control -A _blank )]×100;

In the formula:

IC% - the inhibition rate of triazophos on the antigen-antibody binding reaction;

A _control —the average absorbance value of the negative control well;

A _sample ——the average absorbance value of triazophos standard solution or sample extract in the detection hole;

A _blank —the average absorbance value of the blank control well;

Take the logarithmic value of the concentration of the gradient dilution solution of the triazophos standard sample as the abscissa, and the corresponding percentage of inhibition rate as the ordinate, and draw the standard curve respectively.

The components of the 5-fold PBST solution are: 200 mL, 10% Tween-20: 5 mL, add double-distilled water to make the volume to 1 L; dilute 5-fold PBST with double-distilled water 5 times to obtain the PBST solution.

The preparation of the substrate liquid is: Substrate A: 8.2g anhydrous sodium acetate, 2.5g β-cyclodextrin and 150mg urea hydrogen peroxide, add double distilled water to make up to 1000mL, adjust the pH to 5.0, store at 4°C ; Substrate B: Dissolve 50mg 3,3,5,5-tetramethylbenzidine in 5mL dimethyl sulfoxide, store in the dark at room temperature; 15 minutes before use, mix 14.6mL substrate A and 0.45mL substrate B into a substrate solution.

5. Weigh 1 g of the sample to be tested, crush it, add 5 mL of PBS solution and ultrasonically extract it for 3 times, and filter to obtain the sample extract. Replace the triazophos standard sample gradient dilution solution described in step 4) with the sample extract, repeat step 4) operation, and calculate the content of triazophos in the test substance by the standard curve according to the inhibition rate.

Example 2

This embodiment provides a biomimetic immunoassay method for triazophosphatase labeling, comprising the following steps:

Step 1 is the same as in Example 1.

2. Preparation of enzyme-labeled antigen

Take 0.3 mmol of the hapten, dissolve it in 1 ml of DMF, add 60 μl of n-tributylamine and 30 μl of ethyl chloroformate under stirring, and stir at room temperature for 1 h. Take 300μl of the reaction solution and slowly add it dropwise to 6ml of CBS buffer solution (0.01mol/L) dissolved with 120mgOVA. After stirring the reaction at room temperature, put it into a dialysis bag, first dialyze with distilled water for 3 times, and then dialyze with 0.01mol/L of PBS 3d, change the dialysate 3-4 times a day. Take out and store in a refrigerator at -20°C.

After the antigen is activated, it is coupled to the enzyme protein, and the concentration of the enzyme protein in the reaction solution is 10 mg/ml (the molar ratio of antigen to enzyme protein is 20:1, and the enzyme is horseradish peroxidase). After the reaction, an equal volume of glycerin was added, mixed evenly, aliquoted, and stored at -20°C.

The 5-fold PBS solution is composed of Na ₂ HPO ₄ 12H ₂ O: 68.80g, NaH ₂ PO ₄ 2H ₂ O: 8.97g and NaCl: 45.00g, and distilled water to 1L; Dilute 5 times PBS with double distilled water 5 times to get PBS solution.

3. Preparation of Molecularly Imprinted Polymer Membranes

Triadimefon (dissolved in 30ml of acetonitrile), methacrylic acid and trimethoxypropyl trimethacrylate were mixed according to the molar ratio of 1:5:4, then 60mg of azobisisobutyronitrile was added, and the reaction was stirred for 1.5h. Add 200 μl of the above mixture to each well of a 96-well microplate plate, polymerize at 30°C for 24 hours under vacuum drying conditions, elute with 7:1 (v/v) methanol/glacial acetic acid solution for 15 hours, and elute with methanol solution for 5 hours to remove three Zazolone was dried to obtain molecularly imprinted polymer membranes.

4. Establishment of multi-residue enzyme-labeled biomimetic immunoassay method

The imprinted polymer membrane prepared in step 3 was used as a biomimetic antibody; the enzyme-labeled antigen standard solution prepared in step 2 was diluted 5000 times with PBS solution as the enzyme-labeled diluent.

Set the first row of the 96-well ELISA plate as the blank group, and add only 200 μL of PBS solution to each well; the second row as the control group, add 100 μL of enzyme standard dilution solution and 100 μL of PBS solution to each well; Add triazophos standard solution or sample extract gradient dilution solution and 100 μL enzyme standard dilution solution respectively. After incubation at room temperature for 1 h, the plate was washed five times with phosphate Tween buffered solution PBST. Add 100-200 μL of substrate solution to each well of the 96-well ELISA plate, and react for 30-60 minutes at room temperature. Add 50-100 μL of 1.25mol/L sulfuric acid to each well to terminate the reaction; read the absorbance value A of the 1-8 row of the 96-well microplate plate with a microplate reader, and calculate the inhibition rate respectively;

Calculate the inhibitory rate value of different concentrations of triazophos to the antigen-antibody binding reaction according to the following formula:

IC%=[1-(A _sample -A _blank )÷(A _control -A _blank )]×100;

In the formula:

IC% - the inhibition rate of triazophos on the antigen-antibody binding reaction;

A _control —the average absorbance value of the negative control well;

A _sample ——the average absorbance value of triazophos standard solution or sample extract in the detection hole;

A _blank —the average absorbance value of the blank control well;

Take the logarithmic value of the concentration of the gradient dilution solution of the triazophos standard sample as the abscissa, and the corresponding percentage of inhibition rate as the ordinate, and draw the standard curve respectively.

The components of the 5-fold PBST solution are: 200 mL, 10% Tween-20: 5 mL, add double-distilled water to make the volume to 1 L; dilute 5-fold PBST with double-distilled water 5 times to obtain the PBST solution.

The preparation of the substrate solution is as follows: Substrate A: 8.0g anhydrous sodium acetate, 3.0g β-cyclodextrin and 140mg urea hydrogen peroxide, add double distilled water to make the volume to 1000mL, adjust the pH to 5.2, store at 4°C ; Substrate B: Dissolve 40mg 3,3,5,5-tetramethylbenzidine in 6mL dimethyl sulfoxide, store in the dark at room temperature; 15 minutes before use, mix 14.6mL substrate A and 0.45mL substrate B into a substrate solution.

5. Weigh 2 g of the sample to be tested, crush it, add 10 mL of PBS solution and ultrasonically extract it for 3 times, and filter to obtain the sample extract. Replace the triazophos standard sample gradient dilution solution described in step 4) with the sample extract, repeat step 4) operation, and calculate the content of triazophos in the test substance by the standard curve according to the inhibition rate.

It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.

Claims (10)

1. a kind of bionical enzyme-linked immuno sorbent assay kit of Hostathion, which is characterized in that including：

Molecular imprinted polymer membrane ELISA Plate and Hostathion enzyme-labelled antigen；

It is described to be prepared by the following method to obtain：Using triazolone as template molecule, with function monomer, crosslinking agent, initiator lazy Property solvent in carry out prepolymerization obtain prepolymerization liquid；The prepolymerization liquid is added in ELISA Plate, is eluted after vacuum drying polymerization Template molecule to obtain the final product.

2. kit according to claim 1, which is characterized in that the function monomer is methacrylic acid, the crosslinking Agent is trimethoxy oxypropyl trimethyl acrylate, and the template molecule：Function monomer：The molar ratio of crosslinking agent is 1:5~ 7:2~4；

Preferably, the atent solvent is acetonitrile；It is furthermore preferred that the template molecule：Atent solvent=1mol：20~30ml；

Preferably, the initiator is azodiisobutyronitrile；It is furthermore preferred that the template molecule：Initiator=1mol：40~ 60mg。

3. kit according to claim 2, which is characterized in that the condition of the vacuum drying polymerization is vacuum drying item Lower 30 DEG C~50 DEG C polymerization 12h of part~for 24 hours.

4. kit according to claim 1, which is characterized in that the preparation method of the Hostathion enzyme-labelled antigen includes：

Hostathion haptens is synthesized into envelope antigen using mixed anhydride method, then by the envelope antigen and zymoprotein according to 18 ~22:1 molar ratio is coupled；

Preferably, the zymoprotein is horseradish peroxidase.

5. kit according to claim 4, which is characterized in that described to close Hostathion haptens using mixed anhydride method It is specifically included at the step of envelope antigen：By Hostathion haptens and n,N-Dimethylformamide with 0.2mmol~0.3mmol： The ratio of 1ml is mixed to get mixed liquor, then be added into the mixed liquor positive tri-n-butylamine and ethyl chloroformate reaction 50min~ 70min, then gained reaction solution is mixed with the CBS buffer solutions dissolved with OVA, after dialysis to obtain the final product.

6. according to Claims 1 to 5 any one of them kit, which is characterized in that the kit further includes buffer solution；It is excellent Choosing, the buffer solution is PBS solution.

7. according to Claims 1 to 5 any one of them kit, which is characterized in that the kit further includes substrate solution；

The substrate solution is configured by substrate A and substrate B；

The ingredient of the substrate A includes：The anhydrous sodium acetate of 8.0g/L~8.4g/L, the beta-cyclodextrin of 2.0g/L~3.0g/L with And the carbamide peroxide of 140mg/L~160mg/L, pH=4.8~5.2；

The ingredient of the substrate B includes that ratio is 40mg~60mg:The 3,3,5,5- tetramethyl benzidines of 4ml~6ml:Dimethyl Sulfoxide.

8. a kind of carrying out the bionical MBP enzyme linked immuno-adsorbent assay of Hostathion using claim 1~7 any one of them kit Method, which is characterized in that including：

Blank control wells, negative control hole and detection hole are set on the molecular imprinted polymer membrane ELISA Plate；

Hostathion enzyme-labelled antigen dilution and Hostathion titer or sample extracting solution are added in the detection hole, and different The Hostathion titer or sample extracting solution being added in hole are by gradient dilution；

Hostathion enzyme-labelled antigen dilution and buffer solution are added in the negative control hole；

Wherein, the Hostathion enzyme-labelled antigen dilution is diluted to obtain by Hostathion enzyme-labelled antigen standard solution with buffer solution；

The buffer solution is added in the blank control wells；

Each hole is incubated at room temperature 50min~70min after being added in a manner described, substrate is separately added into each hole after board-washing Liquid terminates reaction after being incubated at room temperature 30min~60min；Microplate reader, which reads each hole absorbance and calculates according to the following formula, to be inhibited Rate：

IC%=[1- (A_Sample-A_Blank)÷(A_Control-A_Blank)]×100；

In formula：

Inhibiting rate of IC% --- the Hostathion to antigen-antibody binding reaction；

A_Control--- the negative control hole mean absorbance values；

A_Sample--- the mean absorbance values of Hostathion titer or sample extracting solution in the detection hole；

A_Blank--- the mean absorbance values of the blank control wells；

Using the logarithm of Hostathion titer gradient dilution liquid or sample extracting solution gradient dilution liquid concentration as abscissa, accordingly Inhibiting rate percentage is ordinate, draws standard curve respectively；

The corresponding absorbance value of the sample extracting solution is substituted into the standard curve to get the sample extracting solution concentration.

9. according to the method described in claim 8, it is characterized in that, the preparation method of the sample extracting solution includes：

Buffer solution ultrasonic extraction is added after sample to be tested is crushed, filters to obtain the final product.

10. according to the method described in claim 9, it is characterized in that, the ratio of the sample to be tested and the buffer solution is 1g ~2g：5ml~10ml.